Impaired bone healing in irradiated patients results from damage to local cell populations and a compromised vasculature. Cell-based therapies represent a powerful alternative to tissue grafts, yet their clinical application requires human cells that can be readily obtained, possess angiogenic potential, and contribute to bone formation upon appropriate stimulation. Endothelial colony forming cells (ECFCs) are a subpopulation of endothelial progenitor cells that exhibit robust angiogenic potential under hypoxic conditions and can form functional vascular networks in vivo. Mesenchymal stem cells (MSCs) are easily retrievable and proliferate rapidly, possess osteogenic potential, and support capillary formation. Successful cell-based therapies for bone healing will be identified by utilizing populations that can synergistically contribute to tissue repair when delivered with a matrix that localizes transplanted populations to the defect site, while also facilitating and enhancing cell survival, proliferation, and differentiation. Fibrin gels mimic the blood clot that is the first step of healing in a natural fracture site and are appealing because they are bioresorbable, injectable, the fibrin can be prepared from the patient's own blood and, like a natural blood clot, they effectively hold cells critical to healing. Chemical and mechanical properties of fibrin gels may be tailored by modifying the relative concentrations of clotting proteins. Stiffer substrates drive bone formation with stem and progenitor cells. As an alternative to high clotting protein concentrations necessary to increase fibrin gel stiffness, we recently developed a method for increasing the stiffness of fibrin gels by manipulating ionic strength. Our central hypothesis is that fibrin gels with enhanced physical properties will be effective vehicles for cotransplanting cell populations that directly participate in neovascularization and bone formation. Aim 1. Determine the vasculogenic and osteogenic potential of ECFCs and MSCs co-cultured within fibrin gels possessing increased stiffness. The ability of engineered fibrin gels to support the vasculogenic and osteogenic response of entrapped cells will be quantified. Assess the ability of ECFCs and MSCs cotransplanted within fibrin gels to enhance vascularization and bone formation in an irradiated rodent calvarial defect model. The contribution of implanted cells toward tissue perfusion and bone formation will be assessed using noninvasive imaging modalities. The proposed research is innovative because it exploits natural participants of tissue healing, fibrin and tissue-forming cells, to overcome tissue microenvironments that inhibit normal repair. We will elucidate the contributions of ECFCs and MSCs when implanted into an irradiated bone defects characteristic of patients with head and neck cancer. Collectively, this research will fill a critical void in our understanding of how vascular and stromal cells promote capillary vessel formation and tissue repair, and these studies have direct relevance for treating nonhealing bone defects, soft tissue defects, and emerging applications in tissue engineering and regenerative medicine. PUBLIC HEALTH RELEVANCE: The development of new cell delivery strategies that accelerate tissue formation and function upon implantation will greatly improve the quality of life for those who suffer from nonhealing tissue defects. We seek to determine if engineered fibrin gels possessing increased stiffness can promote vascularization and bone formation by transplanted vessel- and bone-forming cells.